Recent developments in antibody engineering and recombinant DNA technology have made it possible to generate recombinant antibodies with high specificity and affinity for theoretically any antigen by employing phage display technology and constructing very large repertoires of antibodies that are displayed on the surface of filamentous phage (Winter et. al., (1994) Ann. Rev. Immunol. 12:433-455). International patent application WO 92/18619 describes methods for producing a library of DNA molecules capable of expressing a fusion polypeptide on the surface of a filamentous phage particle (phagemids) and producing heterodimeric receptors such as antibodies, and T-cell receptors.
These large repertoires of naive, immunocompetent, or synthetic antibody fragments are fused to a minor phage coat protein; they are integrated into the DNA of the filamentous phage and displayed on the phage surface. Panning and selection of individual phage clones can screen the phage population containing tens of millions of individual clones through binding to an immobilized antigen (Barbas (1995) Nature Medicine 1:837-839). However, this is a very time-consuming process requiring as much as 6-10 weeks to complete, depending on the complexity of the antigen mixture.
After selection, antibody genes rescued from the phage genome can be expressed very efficiently in bacteria for the production of soluble, functional recombinant antibody fragments (Ward et. al., (1989) Nature 341:544-546). However, the disadvantage of such antibodies is that they are typically naive (i.e., non-immunocompetent) and therefore have a significantly lower binding affinity and are not as efficient or useful for binding experiments. Naïve antibodies are generally regarded as antibodies produced by B-cells that have not undergone class-switching and post-somatic hypermutation in response to exposure to antigen. In most animals, the initial exposure to an unknown antigen results in B-cell production of IgM class of antibodies of relatively low affinity. Subsequent exposure provides a selection process among B-cells whereby a genetic rearrangement occurs within the antibody gene of the corresponding B-cell. The result is a higher binding affinity antibody of a non-IgM class of immunoglobulin such as IgG, IgA, or IgE and their various species-specific subclasses. This in vivo response is critical for obtaining high quality antibodies with a high binding affinity and has yet to be routinely replicated in vitro.
Methods of producing a high affinity, monoclonal antibody to a specific antigen using single human B cells have been described (de Wildt, et al. (1997) J. Immunolog. Meth. 207:61-67; Weitkamp, et al. (2003) J. Immunolog. Meth. 275:223-237; Babcock, et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848). In general, these methods employ sorting methods to select for all antibody-secreting cells using a general cell-surface marker (e.g., CD19) or an enrichment step wherein B-cells are selected for binding to a specific antigen (e.g., U1A protein).
There is a need in the art for a high-throughput approach of producing antibodies with a high affinity and are antigen-specific. Moreover, it would be advantageous for these antibodies to be rapidly produced with minimal or no selection to eliminate the time-consuming processes of panning or screening. The present invention meets this need by providing a one-step selection process in combination with recombinant cell technologies.